Structure of bleomycin-induced DNA double-strand breaks: predominance of blunt ends and single-base 5' extensions

Abstract

In order to examine the structure of bleomycin-induced DNA double-strand breaks, defined-sequence DNA was labeled in each strand at a single restriction site and treated with bleomycin. Various double-stranded fragments results from bleomycin-induced double-strand breaks were isolated, denatured, and run on sequencing gels to determine the sites of cleavage in each strand. For virtually every double-strand break, the cleavage site in one strand was a pyrimidine in a G-Py sequence, reflecting a specificity similar to that of bleomycin-induced single-strand cleavage. However, the cleavage site in the complementary strand were seldom a G-Py sequence, and was usually a site where single-strand cleavage was infrequent. When the sequence at the double-strand break was G-Py-Py'', the break at Py was usually accompanied by a break at the base directly opposite Py, resulting in blunt ends. When the sequence was G-Py-Pu, the break at Py was usually accompanied by a break at the base opposite Pu, resulting in single-base 5'' extensions. Double-strand breaks with 3'' extensions, such as would result from cleavage of two C residues in a self-complementary G-C sequence, were conspicuously absent. These data provide further evidence that bleomycin-induced double-strand breaks do not result from coincidence of independent site-specific single-strand breaks. A model is proposed wherein Fe(III).cntdot.bleomycin and a 4''-peroxyl derivative of deoxyribose, both formed during cleavage at the G-Py site, combine to regenerate activated bleomycin, which then effects a second cleavage at a specific site in the complementary strand, either directly opposite Py or opposite the base one position downstream.